**2. Trypanosomatids**′ **Life Cycle in the Context of In Vitro Screening Assays**

Pathogenic trypanosomatids have complex, digenetic lifecycles, which require the presence of both invertebrate and vertebrate hosts (summarized in Figure 2). In this way, various developmental stages throughout trypanosomatids' lifecycle are required to guarantee their survival and spread.

These diverse stages encompass many metabolic, biochemical, and cell biological adaptations, including a significant variation of cell morphology [23–25]. Because of these changes, it is hard, and sometimes impossible, to establish a correlation between compounds selected in assays targeting different forms of the same parasite (e.g., extracellular vs. intracellular). In the current lack of methodology standardization, this section will discuss the mains aspects to be considered to choose the most adapted in vitro screening assay to start a drug discovery cascade.

Pathogenic trypanosomatids have complex, digenetic lifecycles, which require the presence of

*Genes* **2020**, *11*, 722 3 of 24

owing to climate change, global urbanization, immunosuppressive disease, etc. [15,16].

use and limitations, as well as recent endeavors for discovering lead compounds.

**2. Trypanosomatids′ Life Cycle in the Context of In Vitro Screening Assays** 

Leishmaniasis is estimated to be the ninth largest disease burden among individual infectious diseases, and the most dangerous of the NTDs. Leishmaniasis currently infects around 12 million people worldwide, and it is spreading with ca. 0.7–1 million new cases per year [14]. Dramatically, its visceral form (also referred as VL) has a 95% fatality rate among the poorest people in the world. The control of leishmaniasis relies on old-fashioned, highly toxic chemotherapy using a very limited number of registered molecules (Figure 1). In addition to toxicity, significant drawbacks such as complex route of administration, length of treatment, emergence of drug resistance, and costs limit their use in endemic areas [1,14]. Furthermore, NTDs are becoming emergent diseases in non-tropical countries, triggering vast socioeconomic consequences. The absence of investment to combat NTDs is likely due to their traditional cause of misfortune to poor, rural, and otherwise marginalized populations. However, their impact has shifted because of resistant strains and globalization. Without effective new drugs, the incidence of Chagas disease and leishmaniasis is expected to spread

Traditionally, pharmaceutical companies have shown a very limited interest in improving current therapeutics against trypanosomatid parasites because of the expected low return on investment when targeting communities with little to no purchasing power [17,18]. In order to alleviate the costs and accelerate the marketing process [19–21] (e.g., to avoid obstacles during clinical trials, such as drug toxicity or unfavorable pharmacokinetics) [22], many initiatives are trying to find new indications for already-existing drugs, also known as drug repurposing (or drug repositioning) [1]. On the other hand, other initiatives—especially those stemming from academia—are targeted for identifying new points of intervention and to conceive novel drugs. In both cases, interdisciplinary research between experts in parasitology and chemistry is required, such that the former focus primarily on established drugs to treat infection due to limited access to novel molecules. Markedly, the critical situation with NTDs calls for the urgent development of high-throughput approaches for assessing drug efficacy and resistance, as well as novel therapeutics to avoid the emergence and spread of drug-resistant strains. Through this review, we aim to bring together these two major fields of knowledge and shed some light on the different models that are currently available, in order to build a drug-discovery pipeline targeting trypanosomatids (from in vitro to in vivo approaches), their

**Figure 2.** Life cycles of pathogenic trypanosomatid parasites. The clinically relevant life-cycle stages that are targets for drug intervention are intracellular amastigotes in *Leishmania* sp.; bloodstream **Figure 2.** Life cycles of pathogenic trypanosomatid parasites. The clinically relevant life-cycle stages that are targets for drug intervention are intracellular amastigotes in *Leishmania* sp.; bloodstream forms (bloodstream long slender form (B-LS) and bloodstream short stumpy form (B-SS)) in *Trypanosoma brucei*; and infective trypomastigotes and intracellular amastigotes in *Trypanosoma cruzi*.
